X chromosome inactivation is an essential mammalian epigenetic process that serves to balance the levels of X-linked gene expression between the sexes. The inactive X chromosome (Xi) is the most extensive example of developmentally regulated gene silencing, and provides a model to explore in depth the epigenetic features of heterochromatin and to relate these to the underlying genomic DNA sequence. Unexpectedly, the X-linked macrosatellite DXZ4 is packaged into the opposite chromatin arrangement than that of the surrounding chromosome. Consequently, DXZ4 on the Xi is organized into euchromatin, whereas the array on the active X chromosome (Xa) is heterochromatic. DXZ4 is bi-directionally transcribed on both the Xa and Xi, but the processing of the non-coding RNAs differs between the two chromosomes. Cleavage of the antisense transcript into small RNAs coincides with the heterochromatic form of DXZ4, and the small RNAs themselves directly align with nucleosomes bearing the heterochromatin modification H3K9me3. Meanwhile, antisense RNA originating from the Xi is sufficiently stable that it can be detected as a longer transcript. Intriguingly, the chromatin insulator and epigenetic organizer protein CTCF binds specifically to the Xi array, immediately adjacent to the DXZ4 bi-directional promoter. To what extent CTCF is involved in establishing and maintaining DXZ4 chromatin on the Xi, and the impact this unusual organization has on flanking chromatin is unknown. However, the contrasting arrangement of DXZ4 chromatin between the X chromosomes and the specific binding of CTCF to the Xi is conserved at the functional homologue of DXZ4 on the mouse X chromosome (Dxz4), suggesting that this organization serves an important function. Our long-term goal is to understand the role of DXZ4 on the Xa and Xi and the extent to which it is involved in organizing and maintaining flanking chromatin and gene expression. We hypothesize that (A) The Xa processed antisense small RNAs are intimately linked to H3K9me3, via a mammalian pathway similar to RNAi mediated heterochromatin formation described for S.pombe, and (B) The Xi specific association of CTCF with DXZ4 ensures stabilization of antisense transcripts, packaging of the array into euchromatin and maintenance of flanking heterochromatin. In order to test these hypotheses, we propose three specific aims: (i) To define in detail the chromatin organization of DXZ4/Dxz4 on the Xa and Xi, (ii) To understand the role of DXZ4 features in maintaining chromatin and expression of both the array and flanking genomic interval on the Xa and Xi and (iii) To assess the role of Dxz4 on the mouse X. Taken together, these studies are designed to explore the mechanisms that direct packaging of DNA into euchromatin and heterochromatin and will provide valuable insight into the role of RNA in chromatin organization in complex genomes. As such, this research has direct relevance to all forms of epigenetic abnormalities including imprinting disorders, genetic disease involving mutation of chromatin proteins, and the many chromatin alterations observed in cancer. PUBLIC HEALTH RELEVANCE: The proposed studies aim to investigate an unusual epigenetic phenomena involving the macrosatellite sequence DXZ4 on the inactive X chromosome. DXZ4 is a paradigm to explore in depth the interplay between epigenetic features in establishing and maintaining chromatin organization. Therefore, this research has direct relevance to all forms of epigenetic abnormalities including imprinting disorders such as Prader-Willi/Angleman syndrome or Beckwith-Weidemann syndrome, genetic disease involving mutation of chromatin proteins such as Rett syndrome, and the many chromatin changes observed in cancer. Furthermore, understanding the packaging and organization of DXZ4 will have direct impact on Facioscapulohumeral Muscular Dystrophy research, a devastating macrosatellite contraction disorder.